Assessment of in vivo transfection efficiency of the lead lipoplex system containing aminolipid AL-A12. Evaluation of lipoplex biodistribution and transfection efficiency in vivo in the zebrafish model. Confocal imaging (maximum intensity projections) of transgenic zebrafish embryos with GFP expressing vasculature endothelial cells was performed 4 and 24 h post-injection with fluorescent DOTAP:chol or AL-A12-based lipoplexes entrapping plasmid DNA encoding for RFP. Representative maximum intensity projections of a series of three images are shown (4 h post-injection). Displayed are the merged images, the vasculature expressing GFP (green), the DiD-labeled lipoplexes (blue), and the reporter gene expression (RFP, red). AL-A12 promoted a qualitatively higher gene expression than DOTAP:chol-based lipoplexes h. Scale bars = 80 μm. The right graphs show the quantitative image analysis using Fiji ImageJ of the total area in pixels (top) and the percentage of the area (bottom) of the RFP expression determined 4 and 24 h post-injection of DOTAP:chol (black) and AL-A12-based lipoplexes (gray). The results were not statistically significant at p < 0.05.